Copper-zinc superoxide dismutase (CuZnSOD) is an enzyme found in relatively high concentration in the cytoplasm of all or nearly all eukaryotic cells. It consists of two identical subunits, each containing one Cu2+ and one Zn2+ ion in close proximity, held 6.3 apart by the imidazolate ring of a histidyl residue, which acts as a bridging ligand between the two metal ions. CuZnSOD is one of a class of enzymes known to be excellent catalysts of the disproportionation of superoxide (2 O2 + 2 H+ arrow O2 +H2O2). There is a growing body of evidence indicating that oxidative damage in mammalian cells is associated with aging, carcinogenesis, and induction of other serious disease states and that toxic oxygen metabolites such as superoxide may be responsible for such damage. Superoxide dismutase has been proposed to act as an antioxidant enzyme by lowering the steady state concentration of superoxide in vivo. The overall goals of the research proposed here are (1) to carry out mechanistic and biophysical studies on mutants of the metalloenzyme copper-zinc superoxide dismutase (CuZnSOD) from the yeast Saccharomyces cerevisiae, prepared in our laboratory using site directed mutagenesis, (2) to use the CuZnSOD gene as a starting point for design and synthesis of new metalloproteins and metalloenzymes that are functionally unrelated to CuZnSOD, and (3) to test the new mutant proteins for biological activity. Several classes of new mutant CuZnSODs will be prepared, purified, and fully characterized. One class of mutants will be designed to test our new theory concerning the mechanism of the enzymatic reaction catalyzed by CuZnSOD, i.e., that the active site structure of native CuZNSODs is designed to ensure that the reactivity with superoxide is high but the reactivity with hydroxide is low, and that this arrangement is necessary to maintain the high reactivity of the enzyme at physiological pH. Another class will be new metalloproteins functionally unrelated to CuZnSOD but based on the CuZnSOD protein structure. Such studies are expected to lead to increased insight into the factors that determine the structures and reactivities of naturally occurring metalloproteins. Thirdly, a series of CuZnSOD mutants that have external histidines will be prepared and modified by covalent attachment of a ruthenium complex for determination of intramolecular electron transfer rates over defined pathways. In this manner information will be obtained concerning the role of the cysteine ligand in naturally occurring electron transfer copper proteins. In addition to physical characterization, the mutant SODs will be evaluated for in vivo activity, by constructing and testing strains of yeast containing the site specific mutant CuZnSODs in place of wild type. In addition, genetic methods will be used to select for new active SODs from inactive mutants, complementing our in vitro metalloprotein design efforts. Information gained from this project will add to our understanding of the relationship between physical properties and biological function of CuZnSODs, as well as contribute to a more detailed understanding of metalloprotein structure and function.